FOXL2 mutations in Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome.

Purpose Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) is an autosomal dominant disorder where eyelid malformation associated with (type I) or without (type II) premature ovarian failure (POF). It is ascribed to mutations in the forkhead transcriptional factor2 (FOXL2) gene. The purpose of this study is to identify mutations in FOXL2 of Chinese patients with BPES. Methods Genomic DNA was prepared from leucocytes of peripheral venous blood. The coding regions and nearby intron sequences of FOXL2 were analyzed by cycle and cloning sequencing. Results Four mutations in FOXL2 were identified in six families, including c.241T>C, c.650C>G, c.804dupC, and c.672_701dup. Of the four, the c.241T>C and c.650C>G were novel and would result in missense changes of the encoded proteins, i.e., p.Tyr81His and p.Ser217Cys, respectively. The c.672_701dup (p.Ala224_Ala234dup) was detected in three families, indicating a mutation hotspot. The c.804dupC (p.Gly269ArgfsX265) mutation was found in one family. Conclusions Our results expand the spectrum of FOXL2 mutations and confirm the mutation hotspot in FOXL2.

the FOXL2 consensus sequences from the NCBI Human Genome Database (NC_000003) were imported into the SeqManII program of the Lasergene package (DNAStar Inc., Madison, WI) and then aligned to identify variations. Each mutation was confirmed by bidirectional sequencing. Mutation description followed the nomenclature recommended by the Human Genomic Variation Society (HGVS).
In addition, one variation of insertions with multiple nucleotides that was found in three families was further analyzed by cloning sequencing. PCR products harboring this mutation were subcloned to pMD18-T Simple Vector (TaKaRa BIO, Japan) according to the manufacture's instructions. Clones with the mutant allele as well as the normal allele were selected by using heteroduplex-SSCP analysis. Sequence of the cloned fragment was identified by cycle sequencing as described above. Mutations were confirmed by sequencing three positive clones from each family. One mutation, c.241T>C, was further analyzed by PCR-RFLP analysis since the mutation creates a new FOKI site.

RESULTS
All patients demonstrated typical features of BPES, including small palpebral fissures, ptosis of the eyelids, and epicanthus inversus ( Figure 1). Upon complete sequencing analysis of FOXL2 for 13 probands with BPES, four heterozygous mutations were found in six probands, including c.241T>C, c.650C>G, c.804dupC, and c.672_701dup ( Figure 2; Table  1). Of the four, c.241T>C and c.650C>G are novel. All four heterozygous mutations were further detected by heteroduplex-SSCP analysis, and one (c.241T>C) was further detected by FOKI digestion (Figure 3). These mutations were also present in affected patients from corresponding families but neither in unaffected individuals nor in 100 controls.
The c.241T>C (p.Tyr81His) mutation results in substitution of a charge-free tyrosine with a charge-positive basic hydrophilic histidine within the forkhead domain. The c.650C>G (p.Ser217Cys) mutation is located immediately upstream of the polyalanine domain. The tyrosine at position 81 and the serine at position 217 are well conserved in FOXL2 by ClustalW analysis of 11 orthologs from related vertebrate species ( Figure 4).

DISCUSSION
FOXL2 encodes a forkhead transcription factor containing a forkhead domain for DNA-binding and a polyalanine domain of uncertain function. Strong expression of FOXL2 has been found in eyelids [3,15], developing periocular muscles, and surrounding tissues [16,17]. Of the four mutations identified in this study, the c.241T>C affected the forkhead domain, while the other three (c.650C>G, c.804dupC, and c.672_701dup) were located upstream, within, and downstream of the polyalanine domain, respectively.
Missense mutations in FOXL2 reported so far usually occurred at the forkhead domain [9,[17][18][19], except two, such as c.650C>T in a Belgian family [4] and c.644A>G in a fivegeneration family from south-India [20]. The clinical subtypes of the patients with the c.650C>T and c.644A>G mutations were unknown. The novel c.650C>G (p. Ser217Cys) mutation identified in Chinese family B occurred at the same site as that found in the Belgian family, which is located immediately upstream of the polyalanine domain. The serine at position 217 is well conserved in 11 orthologs (Figure 4). It has been shown that mutations affecting the polyalanine domain induce extensive nuclear and cytoplasmic protein aggregation [21,22]. Missense changes have been suggested to act as null allele leading to BPES phenotype due to haploinsufficiency [4] or dominant-negative effect [20,23].
It has been suggested that FOXL2 mutations truncating the protein led to BPES type I while those extending the mutant protein were associated with type II [3,4]. However, intra-and inter-family phenotypic variations have been found [3,4,19,24,25] so that this genotype-phenotype correlation might not be general [18,19,26]. The c.804dupC mutation has been shown to cause both types of BPES [4,19,25], and the c.672_701dup causing polyalanine expansion most likely leads to BPES type II [19]. Missense mutations have been associated with both BPES type I [17] and II [3,19]. The patients from families A and B in this study, with novel c.241T>C and c.650C>G mutations, respectively, had type II BPES. The c.650C>G mutation is the first mutation described that occurs immediately upstream of the polyalanine domain and associated with type II BPES. This may raise a possibility that the region containing the c.650C>G mutation is of importance for FOXL2 function.
The c.672_701dup (p.Ala224_Ala234dup) was found in families D, E, and F (Table 1), consistent with a mutation hotspot. To check the origin of the c.672_701dup mutation in three families (families D, E, and F in Figure 3), six SNPs

TABLE 2. RESULTS OF SNPS ANALYSIS OF THE THREE FAMILIES WITH THE C.672_701DUP MUTATION
The origin of the c.672_701dup mutation in family D is different from family E as they have different SNP at rs2291252. This mutation in family F is a de novo event, although patient II:1 in family F shares the same haplogroup of the six SNPs with that of family D.
(including rs13325788, rs2291252, rs28937885, rs7432551, rs28937884, and rs11924939) were analyzed ( Table 2). The SNP at rs2291252 is different between patient II:1 from family D and patient III:1 from family E, which may suggest a different origin of the mutant allele. The mutation in family F is most likely a de novo event as BPES was not present in the patients' parents although the SNPs in the patient II:1 in family F were the same as that of II:1 in family D. It has been reported that 30% of the FOXL2 mutations lead to polyalanine expansion [19]. The c.672_701dup has been found in BPES families of Caucasian [4,19,27,28] and Asian origin [10,29].
In summary, we identified two novel and two known mutations in FOXL2 of six Chinese families with BPES. The two novel mutations are the first reported instances that were associated with BPES type II. Our results expanded the spectrum of FOXL2 mutations and confirmed the mutation hotspot in FOXL2.